Hyphal Damage Research Articles

Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Secretory IgA reduced the ergosterol contents of Candida albicans to repress its hyphal growth and virulence

Candida albicans, one of the most prevalent conditional pathogenic fungi, can cause local superficial infections and lethal systemic infections, especially in the immunocompromised population. Secretory immunoglobulin A (sIgA) is an important immune protein regulating the pathogenicity of C. albicans. However, the actions and mechanisms that sIgA exerts directly against C. albicans are still unclear. Here, we investigated that sIgA directs against C. albicans hyphal growth and virulence to oral epithelial cells. Our results indicated that sIgA significantly inhibited C. albicans hyphal growth, adhesion, and damage to oral epithelial cells compared with IgG. According to the transcriptome and RT-PCR analysis, sIgA significantly affected the ergosterol biosynthesis pathway. Furthermore, sIgA significantly reduced the ergosterol levels, while the addition of exogenous ergosterol restored C. albicans hyphal growth and adhesion to oral epithelial cells, indicating that sIgA suppressed the growth of hyphae and the pathogenicity of C. albicans by reducing its ergosterol levels. By employing the key genes mutants (erg11Δ/Δ, erg3Δ/Δ, and erg3Δ/Δ erg11Δ/Δ) from the ergosterol pathway, sIgA lost the hyphal inhibition on these mutants, while sIgA also reduced the inhibitory effects of erg11Δ/Δ and erg3Δ/Δ and lost the inhibition of erg3Δ/Δ erg11Δ/Δ on the adhesion to oral epithelial cells, further proving the hyphal repression of sIgA through the ergosterol pathway. We demonstrated for the first time that sIgA inhibited C. albicans hyphal development and virulence by affecting ergosterol biosynthesis and suggest that ergosterol is a crucial regulator of C. albicans-host cell interactions.Key points• sIgA repressed C. albicans hyphal growth• sIgA inhibited C. albicans virulence to host cells• sIgA affected C. albicans hyphae and virulence by reducing its ergosterol levels

Antifungal mechanism of cell-free supernatant produced by Trichoderma virens and its efficacy for the control of pear Valsa canker.

Pear Valsa canker, caused by Valsa pyri (V. pyri), poses a major threat to pear production. We aimed to assess the effectiveness of the cell-free supernatant (CFS) produced by Trichoderma virens (T. virens) to control the development of pear Valsa canker and reveal the inhibitory mechanism against the pathogenic fungi. Using morphological characteristics and phylogenetic analysis, the pathogen G1H was identified as V. pyri, and the biocontrol fungus WJ561 was identified as Trichoderma virens. CFS derived from WJ561 exhibited strong inhibition of mycelial growth and was capable of reducing the pathogenicity of V. pyri on pear leaves and twigs. Scanning electron microscopy (SEM) observations revealed deformations and shrinkages in the fungal hyphae treated with CFS. The CFS also destroyed the hyphal membranes leading to the leakage of cellular contents and an increase in the malondialdehyde (MDA) content. Additionally, CFS significantly inhibited the activities of catalase (CAT) and superoxide dismutase (SOD), and downregulated the expression of antioxidant defense-related genes in V. pyri, causing the accumulation of reactive oxygen species (ROS). Artesunate, identified as the main component in CFS by liquid chromatograph-mass spectrometry (LC-MS), exhibited antifungal activity against V. pyri. Our findings demonstrate the promising potential of T. virens and its CFS in controlling pear Valsa canker. The primary inhibitory mechanism of CFS involves multiple processes, including membrane damage and negatively affecting enzymatic detoxification pathways, consequently leading to hyphal oxidative damage of V. pyri. This study lays a theoretical foundation for the utilization of T. virens to control V. pyri in practical production.

Discovery of Highly Efficient Novel Antifungal Lead Compounds Targeting Succinate Dehydrogenase: Pyrazole-4-carboxamide Derivatives with an N-Phenyl Substituted Amide Fragment.

Developing environmentally friendly fungicides is crucial to tackle the issue of rising pesticide resistance. In this study, a series of novel pyrazole-4-carboxamide derivatives containing N-phenyl substituted amide fragments were designed and synthesized. The structures of target compounds were confirmed by 1H NMR, 13C NMR, and HRMS, and the crystal structure of the most active compound N-(1-(4-(4-(tert-butyl)benzamido)phenyl)propan-2-yl)-3-(difluoromethyl)-N-methoxy-1-methyl-1H-pyrazole-4-carboxamide (U22) was further determined by X-ray single-crystal diffraction. The bioassay results indicated that the 26 target compounds possessed good in vitro antifungal activity against Sclerotinia sclerotiorum with EC50 values for compounds U12, U13, U15, U16, U18, U22, and U23 being 4.17 ± 0.46, 8.04 ± 0.71, 7.01 ± 0.71, 12.77 ± 1.00, 8.11 ± 0.70, 0.94 ± 0.11, and 9.48 ± 0.83 μg·mL-1, respectively, which were the similar to controls bixafen (6.70 ± 0.47 μg·mL-1), fluxapyroxad (0.71 ± 0.14 μg·mL-1), and pydiflumetofen (0.06 ± 0.01 μg·mL-1). Furthermore, in vivo antifungal activity results against S. sclerotiorum indicated that compounds U12 (80.6%) and U22 (89.9%) possessed excellent preventative efficacy at 200 μg·mL-1, which was the same as the control pydiflumetofen (82.4%). Scanning electron microscopy and transmission electron microscopy studies found that the compound U22 could destroy the hyphal morphology and damage mitochondria, cell membranes, and vacuoles. The results of molecular docking of compound U22 and pydiflumetofen with succinate dehydrogenase (SDH) indicated they interact well with the active site of SDH. This study validated our approach and design strategy to produce compounds with an enhanced biological activity as compared to the parent structure.

Myco-Synthesis of Silver Nanoparticles and Their Bioactive Role against Pathogenic Microbes.

Nanotechnology based on nanoscale materials is rapidly being used in clinical settings, particularly as a new approach for infectious illnesses. Recently, many physical/chemical approaches utilized to produce nanoparticles are expensive and highly unsafe to biological species and ecosystems. This study demonstrated an environmentally friendly mode of producing nanoparticles (NPs) where Fusarium oxysporum has been employed for generation of silver nanoparticles (AgNPs), which were further tested for their antimicrobial potentials against a variety of pathogenic microorganisms. The characterization of NPs was completed by UV-Vis spectroscopy, DLS and TEM, where it has been found that the NPs were mostly globular, with the size range of 50 to 100 nm. The myco-synthesized AgNPs showed prominent antibacterial potency observed as zone of inhibition of 2.6 mm, 1.8 mm, 1.5 mm, and 1.8 mm against Vibrio cholerae, Streptococcus pneumoniae, Klebsiella pneumoniae and Bacillus anthracis, respectively, at 100 µM. Similarly, at 200 µM for A. alternata, A. flavus and Trichoderma have shown zone of inhibition as 2.6 mm, 2.4 mm, and 2.1 mm, respectively. Moreover, SEM analysis of A. alternata confirmed the hyphal damage where the layers of membranes were torn off, and further EDX data analysis showed the presence of silver NPs, which might be responsible for hyphal damage. The potency of NPs may be related with the capping of fungal proteins that are produced extracellularly. Thus, these AgNPs may be used against pathogenic microbes and play a beneficial role against multi-drug resistance.

Cell-free supernatant of Bacillus velezensis suppresses mycelial growth and reduces virulence of Botrytis cinerea by inducing oxidative stress.

As a notorious pathogenic fungus, Botrytis cinerea has been reported to infect more than 1400 species of plants and cause postharvest gray mold of numerous economic fruits, leading to substantial economic losses. Traditional chemical fungicides in pathogen control have potential issues regarding environmental pollution, disease resistance and human health. More safety and efficacious prevention technique of postharvest gray mold are in urgent demand. This study aims to investigate the potential function and mechanism of Bacillus velezensis to control gray mold for harvested fruits. The results showed that the cell-free supernatant (CFS) generated from B. velezensis strain A4 was able to inhibit spore germination, germ tube elongation and hyphal growth of B. cinerea in vitro, and impair the pathogenicity of B. cinerea on the four tested fruits. Further analysis demonstrated that CFS significantly reduced the expression of genes associated with growth and pathogenicity and weakened the ability of B. cinerea spores to penetrate plant cell walls in a dose-dependent manner. Moreover, the CFS destroyed the membrane of hyphae, resulting in exosmosis of cell contents and caused hyphal cells to accumulate excessive reactive oxygen species (ROS), leading to hyphal oxidative damage. Our findings indicate that B. velezensis CFS can damage B. cinerea mycelial cells by promoting excessive accumulation of ROS to realize its biological control function.

Magnesium impairs Candida albicans immune evasion by reduced hyphal damage, enhanced β-glucan exposure and altered vacuole homeostasis.

With a limited arsenal of available antifungal drugs and drug-resistance emergence, strategies that seek to reduce Candida immune evasion and virulence could be a promising alternative option. Harnessing metal homeostasis against C. albicans has gained wide prominence nowadays as a feasible antifungal strategy. Herein, the effect of magnesium (Mg) deprivation on the immune evasion mechanisms of C. albicans is demonstrated. We studied host pathogen interaction by using the THP-1 cell line model and explored the avenue that macrophage-mediated killing was enhanced under Mg deprivation, leading to altered cytokine (TNFα, IL-6 and IL10) production and reduced pyroptosis. Insights into the mechanisms revealed that hyphal damage inside the macrophage was diminished under Mg deprivation. Additionally, Mg deprivation led to cell wall remodelling; leading to enhanced β-1,3-glucan exposure, crucial for immune recognition, along with concomitant alterations in chitin and mannan levels. Furthermore, vacuole homeostasis was disrupted under Mg deprivation, as revealed by abrogated morphology and defective acidification of the vacuole lumen. Together, we demonstrated that Mg deprivation affected immune evasion mechanisms by: reduced hyphal damage, enhanced β-1,3-glucan exposure and altered vacuole functioning. The study establishes that Mg availability is indispensable for successful C. albicans immune evasion and specific Mg dependent pathways could be targeted for therapy.

Antagonizing Impact of Endophytic Fungal Isolates against Potato Black Scurf (Rhizoctonia solani)

Four endophytic fungal isolates, naturally associated with Solanum tuberosum L., were evaluated for their antagonistic activity against Rhizoctonia solani and their ability to suppress black scurf disease of potato tubers. To identify the potential implication of antifungal compounds in their inhibitory activity, cell-free culture filtrates were produced and tested for their antifungal potential against R. solani mycelial growth and for their protective effect against development of on potato tuber slices. Results showed that percent inhibition of R. solani mycelial growth, based on in vitro antibiosis tests, ranged between 16 to 59%. Hyphal damage and lysis were the most frequent stress responses exhibited by the target pathogen during its in vitro interactions with the potato-associated fungi tested. All Cell-free culture filtrates of tested fungi had significantly inhibited the radial growth of Rhizoctonia solani Rs20. The cell-free culture filtrates of Penicillium chrysogenum and Aspergillus niger had decreased pathogen growth by more than 60% over control. These two filtrates (P. chrysogenum and A. niger) were found to be the most effective in decreasing the decay incidence in potato slices by 36 and 40%, respectively, as compared to pathogen-inoculated and untreated control. Thus, the present study clearly demonstrated that fungal isolates, occurring ubiquitously within potato plants, may be explored as potent biocontrol agents against potato-associated fungal pathogens and as source of bioactive metabolites for R. solani suppression.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

Bioinspired green synthesis of silver nanoparticles by using a native Bacillus sp. strain AW1-2: Characterization and antifungal activity against Colletotrichum falcatum Went

Zero-valent silver nanoparticles (ZV-AgNPs) are known as potential antimicrobials and here we report antifungal activity of ZV-AgNPs against Colletotrichum falcatum Went for the first time. ZV-AgNPs were synthesized by using a native Bacillus sp. strain AW1−2, which was identified through 16S rRNA gene sequence analysis. Biogenic ZV-AgNPs were confirmed by monitoring a characteristic absorption peak of UV–vis spectroscopy that was measured at 447 nm. Further, it was found through FTIR and XRD analysis that ZV-Ag nanocrystals were capped with proteins of bacterial origin and their size ranged from 22.33–41.95 nm. The ultrastructure imaging through scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed the morphology of ZV-AgNPs as mono-dispersed spheres and energy dispersive X-ray spectroscopy (EDX) revealed the dominance of silver (84.21 %) in the nano-powder. The ZV-AgNPs significantly inhibited the hyphal growth of Colletotrichum falcatum Went as compared to non-treated control and commercial fungicide both in solid and broth media. The ultrastructure SEM and TEM studies revealed the disrupted hyphal structure and damage to the internal cellular organelles of Colletotrichum falcatum Went treated with 20 μg mL−1 ZV-AgNPs, respectively. It was concluded that green ZV-AgNPs of bacterial origin could be used to formulate a nano-based fungicide to effectively control Colletotrichum falcatum Went, the causal agent of red rot of sugarcane.

Hex1, the Major Component of Woronin Bodies, Is Required for Normal Development, Pathogenicity, and Stress Response in the Plant Pathogenic Fungus Verticillium dahliae.

Woronin bodies are membrane-bound organelles of filamentous ascomycetes that mediate hyphal compartmentalization by plugging septal pores upon hyphal damage. Their major component is the peroxisomal protein Hex1, which has also been implicated in additional cellular processes in fungi. Here, we analyzed the Hex1 homolog of Verticillium dahliae, an important asexual plant pathogen, and we report its pleiotropic involvement in fungal growth, physiology, stress response, and pathogenicity. Alternative splicing of the Vdhex1 gene can lead to the production of two Hex1 isoforms, which are structurally similar to their Neurospora crassa homolog. We show that VdHex1 is targeted to the septum, consistently with its demonstrated function in sealing hyphal compartments to prevent excessive cytoplasmic bleeding upon injury. Furthermore, our investigation provides direct evidence for significant contributions of Hex1 in growth and morphogenesis, as well as in asexual reproduction capacity. We discovered that Hex1 is required both for normal responses to osmotic stress and factors that affect the cell wall and plasma-membrane integrity, and for normal resistance to oxidative stress and reactive oxygen species (ROS) homeostasis. The Vdhex1 mutant exhibited diminished ability to colonize and cause disease on eggplant. Overall, we show that Hex1 has fundamentally important multifaceted roles in the biology of V. dahliae.

Fungal-derived extracts induce resistance against Botrytis cinerea in Arabidopsis thaliana

Development of a sustainable agriculture is a current trend searching for alternative and more environmentally friendly strategies to control crop diseases. The necrotrophic fungus Botrytis cinerea, the causal agent of grey mould, affects crops pre- and post-harvest and cause huge economical losses. Microorganisms and microbially-derived elicitors are known to induce plant defences. Here, we study two fungal extracts derived from Colletotrichum acutatum (isolate M11). The crude extract, called CF (culture filtrate) and the semi-purified fraction ACF (axenic culture filtrate) consist in a blend of fungal-derived elicitors. Their activities were evaluated in the plant-pathogen system Arabidopsis thaliana-B. cinerea. Both CF and ACF induced local and systemic resistance, rendering A. thaliana resistant against grey mould disease. The extracts showed no antifungal activity since no inhibition of mycelium growth, changes in hyphal ultrastructure or damage in cell membrane permeability were observed. These findings would indicate that the protective effect was indirect and it was found that CF and ACF induced up-regulation of the defence-related genes PR1, WRKY70 and ERF6, as well as giving local callose deposition in treated leaves. Both fungal extracts have the potential to be used as a main component of a biologically based disease control product with activity against grey mould, with the advantage that the purification process of CF is simpler than for ACF.

Immunosuppressive Compounds Affect the Fungal Growth and Viability of Defined Aspergillus Species.

Immunosuppressive drugs are administered to a number of patients; e.g., to allogeneic hematopoietic stem cell transplant recipients. Immunosuppressive drugs impair the immune system and thus increase the risk of invasive fungal disease, but may exhibit antifungal activity at the same time. We investigated the impact of various concentrations of three commonly used immunosuppressive compounds—cyclosporin A (CsA), methylprednisolone (mPRED), and mycophenolic acid (MPA)—on the growth and viability of five clinically important Aspergillus species. Methods included disc diffusion, optical density of mycelium, and viability assays such as XTT. MPA and CsA had a species-specific and dose-dependent inhibitory effect on the growth of all Aspergillus spp. tested, although growth inhibition by MPA was highest in A. niger, A. flavus and A. brasiliensis. Both agents exhibited species-specific hyphal damage, which was higher when the immunosuppressants were added to growing conidia than to mycelium. In contrast, mPRED increased the growth of A. niger, but had no major impact on the growth and viability of any of the other Aspergillus species tested. Our findings may help to better understand the interaction of drugs with Aspergillus species and ultimately may have an impact on individualizing immunosuppressive therapy.

Distinct Effects of Immunosuppressive Drugs on the Anti-Aspergillus Activity of Human Natural Killer Cells.

As the prognosis of invasive aspergillosis remains unacceptably poor in patients undergoing hematopoietic stem cell transplantation (HSCT), there is a growing interest in the adoptive transfer of antifungal effector cells, such as Natural Killer (NK) cells. Because immunosuppressive agents are required in most HSCT recipients, knowledge of the impact of these compounds on the antifungal activity of NK cells is a prerequisite for clinical trials. We, therefore, assessed the effect of methylprednisolone (mPRED), cyclosporin A (CsA) and mycophenolic acid (MPA) at different concentrations on proliferation, apoptosis/necrosis, and the direct and indirect anti-Aspergillus activity of human NK cells. Methylprednisolone decreased proliferation and increased apoptosis of NK cells in a significant manner. After seven days, a reduction of viable NK cells was seen for all three immunosuppressants, which was significant for MPA only. Cyclosporin A significantly inhibited the direct hyphal damage by NK cells in a dose-dependent manner. None of the immunosuppressive compounds had a major impact on the measured levels of interferon-γ, granulocyte-macrophage colony-stimulating factor and RANTES (regulated on activation, normal T cell expressed and secreted; CCL5). Our data demonstrate that commonly used immunosuppressive compounds have distinct effects on proliferation, viability and antifungal activity of human NK cells, which should be considered in designing studies on the use of NK cells for adoptive antifungal immunotherapy.

Clinical-scale production of Aspergillus-specific T cells for the treatment of invasive aspergillosis in the immunocompromised host.

Invasive aspergillosis (IA) represents a leading cause of mortality in immunocompromised patients. Although adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) represents a promising therapeutic approach against IA, the complex and costly production limits its broader application. We generated Asp-STs from a single blood draw of healthy individuals or IA patients in only 10 days, by either Aspergillus fumigatus (AF) lysate or peptide stimulation of mononuclear cells. The cells were phenotypically and functionally characterized, and safety was assessed in xenografts. Healthy donor-derived and lysate- or peptide-pulsed Asp-STs presented comparable fold expansion, immunophenotype, and Th1 responses. Upon cross-stimulation, only the lysate-pulsed Asp-STs were empowered to respond to peptide stimulation, although both cell products induced hyphal damage. Importantly, Asp-STs cross-reacted with other fungal species and did not induce alloreactivity in vivo. IA patient-derived T cells displayed an anergic phenotype that prohibited sufficient expansion and yield of meaningful doses of Asp-STs for autologous immunotherapy. Using a rapid and simple process, we generated, from healthy donors but not IA patients, functionally active Asp-STs of broad specificity and at clinically relevant numbers. Such an approach may form the basis for the effective management of IA in the context of allogeneic hematopoietic cell transplantation.

Enhancement of biocontrol potential of biocompatible bovine serum albumin (BSA) based protein nanoparticles loaded bacterial chitinase against major plant pathogenic fungi Alternaria alternata

Abstract Recently, principles of nanoscience and nanotechnology has being embraced in the field of agriculture rather than medicine and health care which has the potential to revolutionize modern day agriculture by effectively controlling insect pests and disease causing phytopathogenic microorganisms. In the present study, biocontrol potential of bovine serum albumin nanoparticles (BSA Nps) loaded extra cellular chitinase (BSA Np-CHS) produced by Serratia marcescens SU05 was studied against phytopathogenic fungi Alternaria alternata. Chitinase was extracted from the chitinase production medium that produced by optimizing various nutrient and process conditions adopting Taguchi method (37 °C temperature, 7 pH, 50 rpm and 1.5 inoculum) shows a extended production of enzyme. Extracted enzyme was purified by ammonium sulphate precipitation and column chromatography separation. Purified enzyme thus obtained was loaded with BSA Nps by cocoeravation method. The method for the preparation of BSA Np-CHS conjugate was optimized by various parameters. Nano enzyme conjugate thus prepared using optimal condition reveals spherical nanosphere with the size range of 110–120 nm and changes in the functional group that was studied by Fourier transform infrared spectroscopy (FTIR) analysis. Biocontrol efficacy of the prepared BSA Np-CHS nano enzyme conjugate against tested fungal strain was studied by determination of fungal biomass and fungal hyphal fragments damage under in vitro condition. BSA Np-CHS nano enzyme conjugate brought about effective reduction of fungal biomass and high rate of fungal hyphae fragments damage in all the tested concentration. Further study will helpful to formulate, apply the nanoformulation under field trails for effective control of phytopathogenic fungi.

Dissimilar pigment regulation in Serpula lacrymans and Paxillus involutus during inter-kingdom interactions.

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming Paxillus involutus, respectively, is induced by complex nutrition, and in the case of S. lacrymans, bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases), but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides, rather than mechanical injury, was the major cause of systemic pigment induction. Conversely, no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid, but not involutin, in return inhibited swarming and colony biofilm spreading of Bacillus subtilis, but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators, signalling pathways and associated transcription factors of basidiomycetes.

Innate and Adaptive Immunity to Mucorales.

Mucormycosis is an invasive fungal infection characterised by rapid filamentous growth, which leads to angioinvasion, thrombosis, and tissue necrosis. The high mortality rates (50–100%) associated with mucormycosis are reflective of not only the aggressive nature of the infection and the poor therapeutics currently employed, but also the failure of the human immune system to successfully clear the infection. Immune effector interaction with Mucorales is influenced by the developmental stage of the mucormycete spore. In a healthy immune environment, resting spores are resistant to phagocytic killing. Contrarily, swollen spores and hyphae are susceptible to damage and degradation by macrophages and neutrophils. Under the effects of immune suppression, the recruitment and efficacy of macrophage and neutrophil activity against mucormycetes is considerably reduced. Following penetration of the endothelial lining, Mucorales encounter platelets. Platelets adhere to both mucormycete spores and hyphae, and exhibit germination suppression and hyphal damage capacity in vitro. Dendritic cells are activated in response to Mucorales hyphae only, and induce adaptive immunity. It is crucial to further knowledge regarding our immune system’s failure to eradicate resting spores under intact immunity and inhibit fungal growth under immunocompromised conditions, in order to understand mucormycosis pathogenicity and enhance therapeutic strategies for mucormycosis.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BackgroundThe human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents.Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections.ResultsAntifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25–64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4–16) for both planktonic and sessile cells.Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms.An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles.ConclusionsThe direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

Methylprednisolone impairs conidial phagocytosis but does not attenuate hyphal damage by neutrophils against Exserohilum rostratum.

Exserohilum rostratum caused a multistate fungal meningitis outbreak following iatrogenic inoculation of contaminated methylprednisolone in the United States. To gain insight into the immunopathogenesis of this infection, we studied the innate host responses of human neutrophils against E. rostratum conidia and hyphae with or without methylprednisolone. The neutrophil-induced percentage fungal damage against conidia and hyphae was effector-to-target ratio dependent (≤55%). While methylprednisolone did not affect neutrophil-induced fungal damage by treatment of Exserohilum or neutrophils, it compromised phagocytosis of conidia (P < 0.05). These findings suggest that methylprednisolone-treated neutrophils may have altered phagocytic clearance of Exserohilum conidia, reducing host capacity to contain the invasive process.